Main text tables and figures.
Table 1. Comparison of Sparkling Lake, Lake Mendota, and Trout Bog. These three lakes were chosen for comparative metatranscriptomics because of their varying trophic statuses, extensive historical data, and previous microbial sampling. Data on surface area, maximum depth, and development on shoreline courtesy of NTL-LTER <lter.limnology.wisc.edu>. Temperature, dissolved oxygen, pH, and conductivity were measured using a HydroLab DS5x Sonde and are averaged over all sampling depths and timepoints for each lake. Chlorophyll and phycocyanin concentrations were measured from the integrated epilimnion samples using a methanol extraction protocol and averaged over all timepoints. Secchi depth was measured at the first timepoint for each lake. Bacterial production was quantified via C14-leucine incorporation and averaged over all timepoints. Due to thunderstorms the night of July 8th, the final 1AM timepoint in Sparkling Lake was collected on July 9th instead.
| Lake Mendota | Trout Bog | Sparkling Lake | |
|---|---|---|---|
| Surface area (km^2) | 39.600 | 0.001 | 0.637 |
| Maximum depth (m) | 25.3 | 7.9 | 20.o |
| Trophic status | Eutrophic | Humic | Oligotrophic |
| Location | Madison, WI USA | Boulder Junction, WI USA | Boulder Junction, WI USA |
| GPS Coordinates | 43.1113, -89.4255 | 46.0412, -89.6861 | 46.0091, -89.6695 |
| Shoreline development | High | Low | Moderate |
| Epilimnion sampling depth | 0-7m | 0-1.5m | 0-4m |
| Temperature (C) | 24.61 | 19.51 | 23.33 |
| Dissolved oxygen (mg/L) | 9.71 | 5.33 | 9.25 |
| pH | 8.64 | 4.03 | 7.52 |
| Conductivity (uS/cm) | 608 | 25.87 | 174.54 |
| Total phosphorus (ug/L) | 18.81 | 23.17 | 6.44 |
| Total nitrogen (ug/L) | 625.83 | 667.98 | 346.46 |
| Total dissolved phosphorus (ug/L) | 8.83 | 15.41 | 5.42 |
| Total dissolved nitrogen (ug/L) | 506.7 | 587.51 | 305.17 |
| Chlorophyll (ug/L) | 6.14 | 14.44 | 1.77 |
| Phycocyanin (ug/L) | 0.74 | 1.94 | 3.15 |
| Bacterial production (cpm) | 60.02 | 30.3 | 3.15 |
| Secchi depth (m) | 4.8 | 1.1 | 6.2 |
| Sampling dates (2016) | July 14-16 | July 8-10 | July 6-9 |
| Sunrise/sunset time | 5:32/20:35 | 5:18/20:49 | 5:17/20:50 |
Table 2. Gene diffs in Lake Mendota
| Number of genes | % Genes more expressed in day | % Genes more expressed at night | % Cyclic genes (12 hr phase) | p-value from t-test of day vs. night read totals | Day/night ratio | |
|---|---|---|---|---|---|---|
| Photosynthesis | 637 | 46.47 | 30.61 | 21.51 | 0 | 2.57 |
| Rhodopsins | 124 | 29.03 | 22.58 | 13.71 | 0.04 | 1.37 |
| RuBisCO | 63 | 23.81 | 4.76 | 0 | 0.67 | 1.15 |
| reductive TCA | 14 | 7.14 | 21.43 | 0 | 0.08 | 1.23 |
| Polyamines | 51 | 0 | 43.14 | 0 | 0.41 | 0.9 |
| Reactive oxygen species | 63 | 39.68 | 3.17 | 15.87 | 0 | 1.76 |
| Protease | 252 | 20.63 | 5.16 | 7.54 | 0.02 | 1.22 |
| Ribose transport | 28 | 0 | 46.43 | 0 | 0.02 | 0.75 |
| General sugar transport | 237 | 44.3 | 33.76 | 5.06 | 0.02 | 1.39 |
| Raffinose/stachyose/melibiose transport | 25 | 0 | 68 | 0 | 0 | 0.58 |
| Glucose/mannose transport | 36 | 0 | 16.67 | 0 | 0.48 | 0.9 |
| Rhamnose transport | 11 | 0 | 54.55 | 0 | 0.16 | 0.84 |
| Xylose transport | 45 | 2.22 | 6.67 | 0 | 0.83 | 0.96 |
| Amino acid transport | 258 | 8.91 | 12.02 | 1.94 | 0.4 | 1.08 |
Table 3. Gene diffs in Trout Bog
| Number of genes | % Genes more expressed in day | % Genes more expressed at night | % Cyclic genes (12 hr phase) | p-value from t-test of day vs. night read totals | Day/night/ratios | |
|---|---|---|---|---|---|---|
| Photosynthesis | 324 | 52.78 | 18.52 | 4.01 | 0.01 | 7 |
| RuBisCO | 59 | 32.2 | 3.39 | 1.69 | 0.01 | 7 |
| Glycoside hydrolases | 13 | 23.08 | 15.38 | 0 | 0.66 | 0.94 |
| Polyamines | 19 | 5.26 | 10.53 | 0 | 0.05 | 0.76 |
| Reactive oxygen species | 58 | 29.31 | 10.34 | 0 | 0.21 | 1.22 |
| Protease | 231 | 18.61 | 12.12 | 0 | 0.05 | 1.56 |
| Ribose transport | 43 | 0 | 46.51 | 0 | 0 | 0.42 |
| General sugar transport | 63 | 6.35 | 49.21 | 3.17 | 0 | 0.55 |
| Xylose transport | 15 | 0 | 33.33 | 0 | 0.02 | 0.58 |
| Methane/ammonia monooxygenase | 24 | 12.5 | 33.33 | 0 | 0.06 | 0.67 |
| Amino acid transport | 101 | 2.97 | 24.75 | 0 | 0.02 | 0.67 |
Table 3. Gene diffs in Sparkling Lake
| Number of genes | % Genes more expressed in day | % Genes more expressed at night | % Cyclic genes (12 hr phase) | p-value from t-test of day vs. night read totals | Day/night ratio | |
|---|---|---|---|---|---|---|
| Photosynthesis | 573 | 30.89 | 10.47 | 16.23 | 0 | 2.76 |
| Rhodopsins | 95 | 6.32 | 1.05 | 2.11 | 0.77 | 0.95 |
| RuBisCO | 97 | 0 | 1.03 | 0 | 0.08 | 0.66 |
| Polyamines | 23 | 4.35 | 13.04 | 0 | 0.18 | 0.8 |
| Alkaline phosphatase | 12 | 0 | 0 | 0 | 0.13 | 0.62 |
| Reactive oxygen species | 68 | 29.41 | 1.47 | 14.71 | 0 | 1.87 |
| Protease | 278 | 10.79 | 0 | 2.16 | 0.14 | 1.46 |
| Carboxylate transport | 27 | 7.41 | 0 | 3.7 | 0.15 | 1.26 |
| Ribose transport | 13 | 0 | 7.69 | 0 | 0.07 | 0.69 |
| General sugar transport | 102 | 6.86 | 3.92 | 1.96 | 0.48 | 0.88 |
| Raffinose/stachyose/melibiose transport | 11 | 0 | 0 | 0 | 0.02 | 0.53 |
| Glucose/mannose transport | 15 | 0 | 0 | 0 | 0.14 | 0.77 |
| Xylose transport | 17 | 0 | 0 | 0 | 0.28 | 0.76 |
| Fructose transport | 13 | 0 | 0 | 0 | 0.09 | 0.69 |
| Amino acid transport | 158 | 3.16 | 0 | 0.63 | 0.31 | 1.18 |
Figure 1. Cyclic trends in Lake Mendota. Cyclic trends with a 12 hour phase were detected in Lake Mendota for several functional categories.
Supp Figure . Top 10 most expressed annotated heterotrophic genes in each study site. Coding regions from reference genomes and metagenome assemblies were clustered at 97% sequence similarity, and the longest coding region was chosen as the representative sequence. Metatranscriptomic reads were mapped to these representative sequences. The top 10 most expressed genes from each lake, filtered to exclude photosynthetic genes, phototrophic organisms, hypothetical genes, and unclassified genes, are presented here. Annotations and classifications are derived from the sequence to which each read mapped.
Supp Figure. Most highly expressed or abundant phyla by lake. To determine which phyla were most abundant or most expressed during our time series, we analyzed metagenomic and metatranscriptomic read counts. The expression of clustered, nonredundant genes was aggregated by phylum and compared to the coverage of those phyla in metagenomes. Genes that could not be classified in a phylum were not included in this analysis. No positive relationship was observed between expression and abundance. Color indicates the type of phylum represented, with grey indicating Archaea, blue for Bacteria, green for Algae, red for multicellular animals, black for protists, and yellow for viruses. The identity of the most expressed and most abundant phyla varied by lake. One phylum, Chloroflexi, was removed from the plot of Lake Mendota due to orders of magnitude higher expression and abundance. This phylum is likely an outlier.
Supp figure. Assessing the variability of metatranscriptomic read counts. One aim of this metatranscriptomic study was to provide information on the variability in gene expression in freshwater that could be used to guide further metatranscriptomic experiments. We calculated the coefficient of variance (CoV) for each gene both within replicates and across samples (A). CoV was lower within replicates than across replicates, indicating that variation across replicates is not technical. High levels of variability were observed in genes from all three lakes. In panel B, three example eigenvectors of genes are shown, including trends for 1) on one day, off the next, 2) up and down across the time series, and 3) observed in only one timepoint.